The primary goal of this research proposal is to characterize a steroid biotransformation in man and animals which is exclusively catalyzed by the host's intestinal bacteria. Recently, Eubacterium sp. #144 and #146 have been isolated and shown to convert 16Alpha-hydroxyprogesterone to 17Alpha-progesterone with the intermediate accumulation of Delta16-progesterone. The biotransformation appears to be catalyzed by two separate enzymes, 16Alpha-hydroxyprogesterone dehydoxylase and Delta16-progesterone reductase. Chemically similar steroid dehydroxylation-reductions catalyzed by other intestinal anaerobes are thought to be carried out by a single enzyme or enzyme-complex. Characterization of this steroid biotransformation will be achieved through the following specific aims: (1) purification and determination of the enzymatic properties of 16Alpha-hydroxyprogesterone dehydroxylase and Delta16-progesterone reductase, (2) determination, by continuous culture, of the effects of growth rate and nutrient limitation on the ability of Eubacterium sp. #144 to catalyze steroid biotransformation, and (3) determination of the structure and metabolism of a novel water-soluble Delta16-progesterone derivative formed spontaneously in the presence of cysteine. This reaction represents a potential metabolic fate for 16Alpha-hydroxyprogesterone mediated by Eubacterium sp. #144 and not previously known.